Upregulation And Activation Of CaSR In PASMCs Contributes To The Development Of Pulmonary Artery Hypertension

PartⅠ Upregulation and activation of CaSR in pulmonary arterysmooth muscle cells from idiopathic pulmonary artery hypertensionObjectives: To explore the expression and function of Ca²⁺-sensing receptor（CaSR）in pulmonary artery smooth muscle cells（PASMCs） from idiopathic pulmonary arteryhypertension（IPAH）,and to evaluate their clinical significance in the pathogenesis ofpulmonary artery hypertension（PAH）.Materials and methods: We measured the expression of CaSR by Westernblot andImmunohistochemical Staining in PAH and non-PAH patients’ lung tissue.Further,wedetermined the the expression of CaSR by Westernblot in PASMCs freshly isolated fromnon-PAH and PAH patients’ pulmonary arteries. Further more, the function of CaSRdetermined by the rise in cytosolic Ca²⁺-concentration ([Ca²⁺]cyt) in PASMCs superfusedwith Ca²⁺-free solution and addition of2.2mmol/L Ca²⁺were measured in PAH and non-PAH patients’ PASMCs.Results: To investigate the potential mechanism of extracellular Ca²⁺-mediatedincrease in [Ca²⁺]cyt, the protein expression level of CaSR in IPAH whole lung tissue,pulmonary arteries（PA） and PASMC is higher than control group.（n=6, P<0.01）.Further,we examined and compared the effects of extracellular Ca²⁺restoration on changesin [Ca²⁺]cytin PASMC from normal subjects and IPAH patients. In normal PASMCsuperfused with Ca²⁺-free solution （plus1mmol/L EGTA; for10minutes）, restoration ofextracellular Ca²⁺（2.2mmol/L） into the bath solution had no effect on [Ca²⁺]cyt. Evenafter long exposure （30and60minutes） of normal PASMC to Ca²⁺-free solution,there wasno change in [Ca²⁺]cytafter restoration of extracellular Ca²⁺（n=56, P>0.05）. In IPAH-PASMC superfused with Ca²⁺-free solution （for10minutes）; however, restorationof extracellular Ca²⁺resulted in a significant increase in [Ca²⁺]cyt（n=55, P<0.05）.Conclusions: The extracellular Ca²⁺-induced increase in [Ca²⁺]cytdue to activationand upregulated CaSR involves in the development of idiopathic pulmonary arteryhypertension.Therefore, CaSR might play a vital role in the pathogenesis of pulmonaryartery hypertension.PartⅡ Activation of CaSR resulted in the extracellular Ca²⁺-inducedincrease in [Ca²⁺]cytvia PLC-IP3pathway in IPAH-PASMCsObjectives: To determine whether PLC-IP3pathway played any role in the course ofthe extracellular Ca²⁺-induced increase in [Ca²⁺]cytby activating CaSR.In order to lay thefoundation for studying the CaSR in the mechanism of pulmonary artery hypertension.Materials and methods: To examine the potential signaling pathway involved in theCaSR-mediated increase in [Ca²⁺]cytin IPAH-PASMC, we performed pharmacologicalexperiments on the extracellular Ca²⁺-mediated increase in [Ca²⁺]cyt. We determinedwhether pretreatment of IPAH-PASMC with the inhibitor of phospholipase C （PLC）,U73122, or the specific blocker of Inositol-2,4,5-Trisphosphate receptor （IP3R）,xestospongin C, blocker of VDCC diltiazem （10μmol/L） or the inhibitor of Na+/Ca²⁺exchanger KB-R7943will inhibited the extracellular Ca²⁺-induced increase in [Ca²⁺]cyt.Results: Short-time pretreatment of IPAH-PASMC with the inhibitor ofphospholipase C （PLC）, U73122（1μmol/L）, or the specific blocker ofInositol-2,4,5-Trisphosphate receptor （IP3R）, xestospongin C （3μmol/L）, significantlyinhibited the extracellular Ca²⁺-induced increase in [Ca²⁺]cyt（P<0.05）. The inactive form ofU73122, U73343（1μmol/L）, however, had no effect on the extracellular Ca²⁺-inducedincrease in [Ca²⁺]cyt. Pretreatment of IPAH-PASMC with the blocker of VDCC,diltiazem（10μmol/L） or the inhibitor of Na+/Ca²⁺exchanger KB-R7943（10μmol/L）, however, hadno effect on the extracellular Ca²⁺-induced increase in [Ca²⁺]cyt（all P>0.05）.Conclusions: These results indicate that activation of PLC and IP3R is involved inthe CaSR-mediated [Ca²⁺]cytincrease in IPAH-PASMC, whereas VDCC and the reversemode of Na+/Ca²⁺exchanger are not involved in the CaSR-mediated Ca²⁺influx or inwardtransportation.Extracellular Ca²⁺-induced [Ca²⁺]cytincrease in IPAH-PASMC is dependentof phospholipase C and the inositol-2,4,5-trisphosphate Receptor. PartⅢ Upregulation and activation of CaSR in pulmonary arterysmooth muscle cells from MCT-PH rats and hypoxia induced PH mice.Objectives: To explore the expression and function of Ca²⁺-sensing receptor（CaSR）in PASMCs from Monocrotaline（MCT）-PH rats and hypoxia induced PH mice,and toevaluate their significance in the pathogenesis of pulmonary artery hypertension in animalmodels.Materials and methods: We measured the expression of CaSR by Westernblot andPCR in whole lung tissue from MCT-PH rats and hypoxia induced PH mice. Further more,the function of CaSR determined by the rise in cytosolic Ca²⁺-concentration ([Ca²⁺]cyt) inPASMCs superfused with Ca²⁺-free solution and addition of2.2mmol/L Ca²⁺weremeasured in primary PASMCs isolated from MCT-PH rats and hypoxia induced PH mice.Results: The protein expression level of CaSR in whole lung tissue and pulmonaryarteries from MCT-PH rats and hypoxia induced PH mice is higher than control group（n=6,P<0.01）. Further,we examined and compared the effects of extracellular Ca²⁺restoration onchanges in [Ca²⁺]cytin PASMC from normal subjects and MCT-PH rats or hypoxiainduced PH mice. In normal PASMC superfused with Ca²⁺-free solution （plus1mmol/LEGTA; for10minutes）, restoration of extracellular Ca²⁺（2.2mmol/L） into the bathsolution had no effect on [Ca²⁺]cyt.[n=45（rats），44（mice）, P>0.05]. In primary PASMCfrom MCT-PH rats or hypoxia induced PH mice superfused with Ca²⁺-free solution （for10minutes）,however, restoration of extracellular Ca²⁺resulted in a significant increase in[Ca²⁺]cyt[n=46（rats），46（mice）, P<0.05].Conclusions: The extracellular Ca²⁺-induced increase in [Ca²⁺]cytdue to activationand upregulated CaSR involves in the mechanism of pulmonary artery hypertension animalmodels.Therefore, CaSR might play a vital role in the development of pulmonary arteryhypertension. Part Ⅳ Inhibition of CaSR prevents and reverses pulmonaryhypertension in rats and mice.Objectives: To explore whether blockade CaSR by the calcilytic NPS2143wilprevent or reverse the development of pulmonary hypertension and right ventricularhypertrophy in rats injected with monocrotaline and mice exposed to hypoxia.Materials and methods: Rats injected with MCT and mice exposed to hypoxia weretwo animal models used in our study. Intraperitoneal injection of the calcilytic NPS2143was given to both control and experimental PH models from Days14to24.Hemodynamics, right heart hypertrophy （RV/LV+S ratio）, and right ventricle myocardiumfibrosis were measured and compared in control animals, experiment PH animals, and PHanimals treated with NPS2143.Results: To test the in vivo therapeutic effect of the CaSR antagonist, we examinedand compared the right ventricular systolic pressure （RVSP）, the ratio of right ventricle/leftventricle+septum[RV/（LV+S）], and muscularization of distal pulmonary arteries innormotensive control （Norm） rats（n=6） and MCT-injected rats with and without treatmentwith NPS2143, a CaSR antagonist（n=6each）. Injection of MCT （60mg/kg） in ratssignificantly increased RVSP and caused right ventricular （RV） hypertrophy comparedwith the normotensive control （Norm） rats injected with vehicle （DMSO）. Intraperitonealinjection of NPS2143（4.5mg/kg per day） had little effect on RVSP and RV/（LV+S） ratioin Norm rats （P>0.05）but significantly attenuated the increase in RVSP and theRV/（LV+S） in MCT-PH rats （all P<0.05）.The MCT-induced increases in RVSP and RVhypertrophy were associated with significant pulmonary vascular remodeling; the vascularmedial wall thickness of small pulmonary arteries with the outer diameter less than100μmwas significantly greater in MCT-injected rats than in Norm rats （all P<0.05）. Treatmentwith the CaSR antagonist （NPS2143） significantly prevent and reversed themuscularization of small pulmonary arteries. To further validate the pathogenic role ofupregulated CaSR in the development of pulmonary hypertension and the therapeuticeffect of CaSR antagonists on experimental pulmonary hypertension, we repeated theexperiments mentioned above in the HPH mouse model. Intraperitoneal injection of NPS2143had little effect on RVSP and RV/（LV+S） ratio in normoxic control mice （Nor）（n=6）（all P>0.05）but significantly inhibited the increase in RVSP and RV/（LV+S） ratio in hypoxic mice （Hyp）（n=6）（all P<0.05）.Furthermore, inhibition of CaSR also significantlyinhibited the hypoxia mediated pulmonary arterial wall thickening and reversed thehypoxia-induced decrease in branch and junction numbers （and total length） of smallpulmonary arterial trees （all P<0.05）.Conclusions: These data imply that intraperitoneal injection of the CaSR antagonistis an efficient therapeutic approach to inhibit the development and progression ofpulmonary vascular remodeling and right ventricular hypertrophy in animal models withexperimental pulmonary hypertension induced by injection of monocrotaline and exposureto hypoxia. The observations from this study strongly indicate that increased expressionand function of CaSR may play a pathogenic role in the development of pulmonaryvascular remodeling and antagonists of CaSR, or calcilytics, may have great therapeuticpotential for patients with pulmonary arterial hypertension.